Polycystin-2 is a non-selective calcium channel that regulates tubulogenesis and maintains homeostasis in several organ systems, including the liver, heart, and kidneys. Mutations in polycystin-2 account for 15% of the cases of people with autosomal dominant polycystic kidney disease (ADPKD), a genetically inherited disorder causing renal failure. Most polycystin-2 in epithelial cells localizes to the endoplasmic reticulum (ER) where it combines with the IPS receptor to control calcium homeostasis. Yet little is known about the mechanisms that localize this polytopic membrane channel to the ER. Our laboratory identified PACS-2 as a novel sorting protein and recently discovered that it binds to the cytosolic domain of polycystin-2 and is required to localize the channel to the ER. PACS-2 connects polycystin-2 to COPI, a vesicular coat protein that controls Golgi-to-ER retrieval. PACS-2/COPI may control the ER localization of polycystin-2 by directing an efficient Golgi-to-ER trafficking step. To test this hypothesis, experiments in Aim 1 will determine whether ER localization of polycystin-2 requires COPI and whether PKD2 is localized to the ER through a retentionor retrieval-based mechanism. To test the role of COPI in controlling the ER localization of full-length polycystin-2, we will use siRNA depletion of the beta-COP subunit and develop two polycystin-2 reporter constructs will be developed to determine quantitatively whether polycystin-2 is localized to the ER by a Golgi-to-ER retrieval step and the role of PACS-2 and COPI in directing this pathway. Specific aim 2 will determine which of the seven COPI subunits and residues in the polycystin-2 cytosolic domain-in addition to phosphorylated Ser812-are important for binding to PACS-2 and directing ER localization of polycystin-2. Yeast two-hybrid genetic screens and protein binding assays will identify COPI subunits and amino acid residues within the polycystin-2 cytosolic domain essential for complex formation. Polycystin-2 reporter molecules containing mutations of identified amino acids will be expressed to rigorously determine their role in the ER localization of polycystin-2 and their potential role in the targeting of polycystin-2 to the TGN, cell surface and primary cilia. Together, these studies will identify the fundamental mechanisms that control the subcellular localization of polycystin-2 and may provide a foundation to understand how intracellular signaling pathways are disrupted in polycystic kidney disease. The proposed studies are relevant to public health because insight into how polycystin-2 action is regulated is crucial for understanding kidney function and for developing therapies that can protect ADPKD patients from the devastating effect of this disease. [unreadable] [unreadable] [unreadable] [unreadable]